JPS5951356A - Multilayer assay element for detection of protein - Google Patents

Abstract

PURPOSE:To achieve a highly sensitive determination of protein by arranging water soluble cupric salt, a copper chelating agent and a alkaline mixture of more than one kind of alkaline metal hydroxides or alkaline metals or alkali earth metal salts to be contained in a reagent layer or a porous development layer thereon. CONSTITUTION:A water soluble cupric salt such as CuSO4, a copper chelating agent such as tartaric acid or salts thereof, an alkaline mixture of more than one kind of compounds selected from alkaline metal hydroxides such as KOH and LiOH or alkaline metals or organic and inorganic salts thereof are arranged to make a protein detection reagent, which is contained in a reagent layer formed on a non-liquid permeable and light transmitting support or a porous development layer thereon to produce a multilayer assay element. An assay element preservable for a long time at the normal temperature is obtained which provides a coloring of a high density with limited sample liquid while capable of quick determination of protein.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to analytical chemistry, and more particularly to analytical elements for analyzing proteins in fluid samples.

In recent years, in the field of analytical chemistry, dry multilayer analytical elements have been developed that have significantly improved quantitative performance and operability compared to methods in which analytical reactions are carried out in a solution system. for example,
As described in US Pat. No. 3,992,158, a porous spreading layer containing all the reagents necessary for analysis in advance is laminated on a reagent layer of a hydrophilic colloid material.

However, it is intended to be used in the field of clinical chemistry,
It is disclosed that the pH conditions are relatively mild, neutral, weakly alkaline, and weakly acidic.

However, in the field of analytical chemistry, various useful reactions are known that allow analytical reactions to be carried out under severe conditions, such as under strong alkalinity.

For example, in JP-A-54-101398, high p
An element for the analysis of liquids under conditions i.

The above patent states that the combination of an alkali substantially free of sodium ions and an alkali-protecting polymer constitutes a stable layer. However, the alkali-protecting polymers used in these elements cannot sufficiently protect alkalis, and when stored for a long period of time, they deteriorate due to moisture in the air, carbon dioxide, etc., resulting in a decrease in analytical sensitivity. have.

Furthermore, the analytical element disclosed in the above-mentioned patent has a serious drawback in that the color density is low, which significantly impairs quantitative performance.

An object of the present invention is to eliminate the above-mentioned drawbacks and provide an element capable of analysis under strong alkalinity.

As a result of extensive research in line with the above objectives, the present inventor has found that
a liquid-impermeable, light-transparent support; at least one reagent layer containing at least one reagent that reacts with a component in a fluid sample located on one side of the support; and the support of the reagent layer. and at least one porous development layer located on the opposite side of the reagent layer and the porous development layer, at least one of the reagent layer and the porous development layer contains a water-soluble cupric salt, a copper chelating agent, The above object can be achieved by a multilayer analytical element for protein detection, which is characterized by containing an alkaline mixture of at least two selected from alkali metal hydroxides and alkali metal or alkaline earth metal salts. Ta. In an embodiment of the present invention, it is preferable to use steel sulfate as the water-soluble cupric salt and tartaric acid or a salt thereof as the chelating agent.

Hereinafter, the analytical element of the present invention will be explained in more detail.

The reagent layer according to the present invention is formed by coating a support with at least one layer of a binder containing at least one type of reagent that reacts with the components in the fluid sample r1. In addition, a powder filter paper or a fibrous material whose length has been adjusted by beating the binder on its surface,
Alternatively, a layer having interconnecting voids may be formed by coating microscopic polymer beads.

The porous spreading layer according to the present invention is a protein macromolecule formed by coating at least one layer of fibrous or polymeric microbeads similar to those used in the reagent layer carrying a binder on the reagent layer. It is a layer with interconnecting voids through which the pores flow.

The cupric salt, copper chelating agent, and alkaline mixture according to the present invention may be contained alone or in a mixture in the reagent layer and the porous development layer.

The other reagent layer according to the invention, which does not contain an alkaline mixture, is preferably composed of a hydrophilic colloid material.

Depending on the characteristics of the reagent used, the reagent can be contained in the reagent layer by a dispersion method such as an oil-protect dispersion method or a direct dispersion method, or by dissolution, which are known in the Imashin industry.

As the cupric salt according to the present invention, water-soluble organic or inorganic cupric salts such as copper chloride, steel bromide, copper acetate, copper nitrate, copper sulfate, etc. are used. Copper sulfate is preferred, as it can maintain guaranteed purity.

As the copper chelating agent, ethylenediamine, nitriloacetic acid, citric acid, tartaric acid, and salts thereof can be used, but tartaric acid or a salt thereof is preferred from the viewpoint of protein color development.

The alkali metal hydroxides according to the present invention include sodium hydroxide, potassium hydroxide, and lithium hydroxide, and the alkali metal or alkaline earth metal salts include sodium, potassium, lithium, calcium, magnesium, barium, carbonates, sulfates of
Examples include nitrates, phosphates, borates, acetates, chlorides, bromides, iodides, fluorides, and the like.

In the present invention, containing two or more of the above compounds means that in the case of a combination of alkali metal hydroxides,
Any proportion may be used, but at least 1% of any one of them should be contained. In the case of a combination of an alkali metal hydroxide and an alkali metal or alkaline earth metal salt, the proportion of the alkali metal hydroxide is 99-40%, preferably 95-50%.

The alkaline mixture can be contained in various concentrations relative to the binder, but it is not more than 100 times the weight, preferably not more than 70 times the weight.

6- The alkaline mixture, water-soluble cupric salt, and copper chelating agent according to the present invention may be added alone to at least one of the reagent layer and the developing layer of the analytical element of the present invention, or as a mixture in the same layer. For example, it can be added to an organic solvent or aqueous solution containing a binder, dispersed or dissolved, and then applied as a desired layer. Especially when containing an alkaline mixture in the reagent layer,
Depending on the purpose and effect, it can be placed in the same layer as other reagents or in a separate layer.

It also has the function of accommodating macromolecules such as proteins, Japanese Patent Application No. 56-155788 and No. 57-6505.
It is also possible to apply the reagent layer with interconnecting voids as described in No.

In addition, as a porous development layer, for example, Japanese Patent Publication No. 53-216
No. 77, JP-A-55-164356, patent application 1987-1
No. 3203, No. 56-65446, No. 56-1897
It is possible to use those described in No. 84, etc., and it is preferable to use a spreading layer whose surface is composed of polymer micro beads or fibers with complementary openings.

The binder used in the present invention includes polymeric substances soluble in organic solvents, such as polystyrenes, polyacrylic esters, polymethacrylic esters, alkyl celluloses such as methyl cellulose and ethyl cellulose, polyvinyl butyral, polyvinyl carbonate, etc. Or water-soluble polymer substances, such as water-soluble cell 0-28 bodies such as carboxymethyl cellulose and hydroxyethyl cellulose, pullulan, pullulan derivatives such as carboxymethyl pullulan, water-soluble vinyl polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. etc.

Furthermore, water-soluble polyamides and the like can also be used, and the above-mentioned water-soluble monomers can also be used in various ways.

Examples include vinyl acid amides such as acrylamide and methacrylamide, and vinyl heterocyclic rings such as N-vinylpyrrolidone and N-vinylimidazole.

It is possible to mix two or more types of the above-mentioned binders depending on the purpose, and other vinyl compounds can also be copolymerized as long as the purpose does not deviate from the purpose.

81- Furthermore, various additives such as preservatives, buffering agents, surfactants, etc., which may be used additionally in the present invention, may be added as desired.

In particular, surfactants can be effectively used to adjust the permeation rate when fluid sample I''l is applied to the device of the present invention.

As a surfactant with a usage ratio of P, it is possible to use surfactants regardless of whether they are ionic (anionic or cationic) or nonionic, but preferably nonionic surfactants are effective. be. Examples of nonionic surfactants include polyalkylene gelicol derivatives of alkyl-substituted phenols such as 2,5-di-1-butylphenoxypolyethylene glycol, p-octylphenoxypolyethylene glycol, and p-isononylphenoxypolyethylene glycol; Examples include polyalkylene glycol esters of higher fatty acids. These surfactants have the effect of regulating the rate of penetration of the fluid sample into the reagent layer and at the same time suppressing undesirable chromatographic phenomena.

9- The above surfactants can be used in widely selected amounts, but range from 10% to 0.005% by weight, preferably from 6% to 0.00% by weight, based on the weight of the coating fluid. 05 weight percent can be used.

The liquid-impermeable, light-transparent support (hereinafter referred to as the support of the present invention) used in the analytical element of the present invention may be of any type as long as it is liquid-impermeable and light-transparent. However, various polymeric materials are suitable for this purpose, such as, for example, cellulose acetate, polyethylene terephthalate, polycarbonate, or polystyrene. The thickness of the support in this case is arbitrary, but is preferably about 50 to 250 microns. Further, one side surface of the support according to the present invention on the observation side can be arbitrarily processed depending on the purpose. Next, when the reagent layer according to the present invention is provided on the above support, it can be directly coated, but in some cases, a light-transparent undercoat layer may be used to bond the reagent layer and the support. Improving sexuality is effective.

The analytical element of the present invention can take any one of a variety of different configurations. Furthermore, the reagent layer and various functional layers, reagent-containing layers, and members of the present invention, such as U.S. Pat.
, 992, 158, a reagent layer, a reflective layer, an undercoat layer,
Radiation blocking layer as described in U.S. Pat. No. 4,042,335, Barrier layer as described in U.S. Pat. No. 4,066,4.03, Registration layer as described in U.S. Pat. No. 4,144,306, U.S. Pat. , 166.093, U.S. Pat. No. 4,127,4
Scintillation layer described in No. 99, JP-A-55-908
59 and U.S. Pat. No. 4,110,079
It is possible to construct an analytical element suitable for the purpose of the present invention by arbitrarily combining the breakable bod-like members described in the above-mentioned issues.

The above-mentioned layer can be formed into a layer having an arbitrary thickness by using a slide hopper coating method, an extrusion coating method, a dip coating method, etc., which are conventionally known in the photographic industry.

After obtaining an analytical result as a detectable change using the analytical element of the present invention, it is measured by reflection spectrophotometry. The amount of the unknown test substance can be determined by applying the measured value thus obtained to a calibration curve prepared in advance.

The analytical element of the present invention configured as described above can achieve its purpose by supplying a fluid sample from the spreading layer and observing the analytical reaction in the reagent layer from the transparent support side.

The amount of fluid sample applied to the analytical element of the present invention can be determined arbitrarily, but is preferably from about .mu.l to about 5 .mu.l.
μl, more preferably about 5 μl to about 5 μl. It is usually preferred to apply a fluid sample of about 10 microns.

The analytical element of the present invention is suitable for performing the Buret reaction for detecting proteins under highly alkaline conditions, and has excellent color development, storage stability, and quantitative performance.

The present invention will be explained in more detail below using Examples, but the present invention is not limited thereto.

Example-1 12- A multilayer analytical element of the present invention was prepared by sequentially coating layers having the compositions shown in Table 1 on a transparent, primed polyethylene terephthalate support having a film thickness of about 180 microns.

13- Multilayer analytical elements having the above configuration were created, and each was designated as analytical elements (1) to (7) of the present invention.

Furthermore, for comparison, a multilayer analytical element was prepared by coating a polyethylene terephthalate film support with a reagent layer containing the alkali-protecting polymers agarose, copper sulfate, and tartaric acid, and a developing layer consisting of microcrystalline cellulose particles. It was made into a multilayer analytical element.

The above-mentioned multilayer analytical element of the present invention and comparative multilayer analytical element were prepared immediately after the above-mentioned element manufacture and those stored at 5° C. and 55% RH for ω days, and bovine serum albumin levels of 2.5, 7.
10 g of each aqueous solution was dropped onto 10 μl of the developing layer, and 3
After incubation at 7°C for 7 minutes, the reflection density was measured from the support side at 540 nm. The results are shown in Table 2 below.

7. Ranno 14-15- As is clear from Table 2 above, the analytical element of the present invention exhibits better color density than the known comparative analytical element, and
It can be seen that this is a good multilayer analytical element that shows almost no decrease in sensitivity even after long-term storage.

Example 2 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the following composition on a transparent undercoated polyethylene terephthalate support having a thickness of about 180 microns.

Multilayer analytical elements having the above configuration were created, and each was designated as analytical elements (8) to (10) of the present invention.

As in Example-1, the above device immediately after manufacturing and at 5°C.
, 55% volumes were stored between Sekiguchi and bovine serum albumin was 2, 5, 10. ! Each aqueous solution of lit/dl was dropped onto a 10 μm developing layer, incubated at 37° C. for 7 minutes, and then the reflection density was measured from the support side at 540 nm. The results are shown in Table 4 below.

As is clear from the results in Table 4 above, the analytical element of the present invention has the same properties as shown in Example 1 even when only a hydrophobic polymer substance (reagent dispersion coating) is used as a binder in the reagent layer. It can be seen that it is a good multilayer analytical element that exhibits good color density like the multilayer analytical element and shows almost no decrease in sensitivity even after long-term storage.

Example 3 A multilayer analytical element of the present invention was prepared by sequentially coating layers having the following composition on a transparent undercoated polyethylene terephthalate support having a thickness of about 180 microns.

19- Multilayer analytical elements having the above configuration were created, and each was used as the analytical element (1, 1) of the present invention (13).

As in Example-1, the above device immediately after manufacturing and at 5°C.
, 55% volumes stored for (a) days were prepared, and 1 aqueous solution containing bovine serum albumin of 2, 5, and 10 g/d/ was prepared.
0μ! After dropping it onto the developing layer and incubating at 37°C for 7 minutes, the reflection density was measured from the support side at 540 nm.

The results are shown in Table 6 below.

As is clear from the results in Table 6 above, the analytical element of the present invention exhibits good color development even when a reagent layer having interconnected voids made of polyvinyl alcohol/styrene polymer particles is applied. It can be seen that it is a multilayer analytical element that shows the concentration and shows almost no decrease in sensitivity even after long-term storage.

Agent: Kuwa Hara Yoshimi 21-

Claims (1)

[Scope of Claims] 1. A liquid-impermeable, light-transparent support, and at least one reagent layer located on one side of the support and containing at least one reagent that reacts with components in a fluid sample. , and at least one porous development layer located on the opposite side of the support to the reagent layer, in which at least one of the reagent layer and the porous development layer contains a water-soluble powder. A multilayer analytical element for protein detection characterized by containing a dicopper salt, a copper chelating agent, and an alkaline mixture of at least two selected from alkali metal hydroxides or alkali metal or alkaline earth metal salts. . 2. The multilayer analytical element for protein detection according to claim 1, wherein the water-soluble cupric salt is steel sulfate, and the steel chelating F agent is tartaric acid or a salt thereof. −1=